Project Abstract Although the gut microbiome is an independent environmental contributor to both obesity and diabetes, the biochemical mechanism(s) underlying the microbiome?s effects on these diseases are unclear. Mouse models have suggested that dysbiosis may affect host metabolism through altered bile acid (BA) signaling. Recent studies have shown that BA signaling is necessary for microbiota-induced dysmetabolism and that it plays an important role in the gut?s control of glucose homeostasis. Therefore, manipulation of BA signaling by the gut microbiome may have therapeutic potential in obesity, type 2 diabetes, and non-alcoholic fatty liver disease. One method to understand the functional role of the microbiome is to use engineered probiotics (EnProbs), commensal bacteria that can colonize the gut of the host and can be genetically manipulated to express enzymes of interest. The primary hypothesis of this proposal is that manipulation of the gut microbiome with EnProbs will help determine the functional role of gut microbiota in BA signaling and host metabolism. Three biochemical transformations will be investigated as part of this proposal: deconjugation by bile salt hydrolase (BSH), 3? sulfonation by SULT2A1, and 7?-dehydroxylation (7DH) by a microbial multigene operon. Hydrolysis of the BA side chain amino acid by BSH is the first step toward secondary BA metabolism. Sulfonation of BAs by BAS blocks reabsorption from the lumen. In metatranscriptomic data of the gut lumen, the 7DH pathway was overrepresented in an obesity-protective feeding paradigm. Preliminary results show that EnProbs marked with fluorescence and expressing BSH can colonize wild-type C57Bl/6 mice for more than a week after a 48 hour gavage. In this proposal, a series of EnProbs expressing BSH, SULT2A1, and a 7DH operon or their catalytically- inactive controls will be introduced into healthy and disbiotic mice. First, the response of the gut microbiome composition and luminal metabolites to EnProb colonization in wild-type mice will be assessed by 16S sequencing and LC-MS. Enrichment with specific enzyme activity should produce corresponding biochemical changes in the luminal metabolite pool, but subsequent effects on the luminal microbial ecology and metabolites are unknown. Second, the effects of the enzymatically-active BSH EnProbs on host BA signaling, insulin sensitivity, lipid and glucose metabolism, and whole-body metabolism will be assessed using the diet-induced obesity mouse model and leptin-knockout (ob/ob) mice. Developing and using EnProbs to identify the role of the gut microbiome expands our ability to (1) understand the role of the gut microbiome in metabolism and (2) manipulate the function of the gut microbiome to achieve a health benefit.